Project Details
Projekt
In vivo profiling of authentic cytosolic targeting routes of mitochondrial precursor proteins
Subject Area
Biochemistry
Term
since 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 541620165
The vast majority of the mitochondrial proteome is synthesized in the cytosol and imported into mitochondria by translocases in the outer and inner mitochondrial membrane. While it is assumed that protein import into mitochondria is predominantly post-translational, recent findings imply that mitochondrial precursors can also be targeted co-translationally or use ER surface mediated import to reach their target organelle. However, the substrate spectra of the individual import routes and the involved cytosolic targeting factors for all three pathways remain largely elusive. We will combine the unique expertise of the Kramer lab in the analysis of translation and co-translational protein maturation using ribosome profiling based techniques and the Vögtle lab in mitochondrial precursor targeting and mitochondrial stress responses to elucidate the initial cytosolic events in mitochondrial protein biogenesis. For this we will employ and further develop selective and compartment-specific ribosome profiling (RP) to investigate the authentic import pathways of mitochondrial proteins in the model organism S. cerevisiae in vivo. This will be extended by total translatome analyses combining RP with RNAseq using an established mtUPR model to reveal the dynamic modulation of mitochondrial protein targeting under mitochondrial stress conditions. Finally, we will use proteomic and selective RP approaches to uncover cytosolic targeting factors and components of the proteostasis network that characterize and thus define the different import pathways. Our analyses will allow simultaneous monitoring of all cytosolic import routes into mitochondria in vivo and identify the bona fide substrate spectrum of each biogenesis pathway. We will investigate the modulation of cytosolic targeting processes upon mitochondrial stress and identify the targeting factors and chaperones that perform cytosolic triage of mitochondrial precursors to prevent their toxic accumulation and overloading of the cytosolic proteostasis network. We believe that selective and compartment-specific ribosome profiling is a highly valuable key technique to address the research questions of this SPP initiative in a global and unbiased manner. We expect that the research consortium will greatly benefit from our comprehensive and in-depth data analyses of the mitochondrial import pathways on a global scale, including the binding profiles of targeting factors and cytosolic chaperones.
DFG Programme
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